Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible ...states including quiescent, inflammatory, and myofibroblastic phenotypes; however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology.
To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetic depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy, various coculture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition, and tumor dissemination and metastasis.
Our in vivo findings showed that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, coculture experiments of tumor organoids and CAFs showed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis showed that patients with pancreatic cancer with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC.
Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work shows that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
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One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for ...endoscopic ultrasound-guided fine-needle aspiration biopsies, typically resulting in specimens with limited tumor cell yield. Here, we tested conditioned media of individual PDOs for cell-free DNA to detect driver mutations already early on during the expansion process to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72 hours after biopsy, recapitulate the mutational profile of the primary tumor, indicating suitability of this approach to subject PDOs to drug testing in a reduced time frame. In addition, we demonstrated that this workflow was practicable, even in patients for whom the amount of tumor material was not sufficient for molecular characterization by established means. Together, our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach, this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.
Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma which plays a critical role in the progression and therapeutic resistance of ...PDAC. The stroma is composed of extracellular matrix proteins, mainly deposited by the cancer-associated-fibroblasts (CAFs) and various types of immune cells. Cancer-associated fibroblasts display a high degree of interconvertible states including quiescent, inflammatory and myofibroblastic phenotypes. However, the mechanisms by which this plasticity is achieved are poorly understood. Here, we demonstrate that CAF plasticity promotes PDAC cell aggressiveness through multiple mechanism, particularly promoting Epithelial-to-Mesenchymal Transition and immune cell infiltration.
Methods: To manipulate fibroblast plasticity in PDAC, we generated genetically engineered mouse models (GEMMs) in which CAF plasticity is modulated by genetical depletion of the transcription factor Prrx1 in fibroblasts by using orthotopic implantation models (Sm22-CreERT, Prrx1fl/fl, Rosa26mTmG) as well as dual recombinase-driven GEMMs (Pdx-Flp, FSF-KrasG12D/w t, p53fr/wtt, Sm22-CreERT, Prrx1fl/fl). To characterize the impact of CAFs on tumor differentiation, immune cell infiltration and response to chemotherapy various in vivo and in vitro co-culture experiments were performed.
Results: Our in vivo results demonstrate that restraining CAF plasticity by Prrx1-depletion leads to more differentiated tumors, disrupts systemic tumor dissemination, including circulating tumor cells as well as metastases. Interestingly in tumors with Prrx1-deficient stroma, infiltration of macrophages and lymphocytes was increased. Specifically, we observed more B-cells as well as cytotoxic T-cells. Gene expression profiling of primary murine fibroblast samples revealed that Prrx1-deficient CAFs express myofibroblastic gene signatures characterized by ECM secretion phenotype. Indeed, on a functional level Prrx1-deficient CAFs secret more collagen and are highly migratory. Additionally, co-culture experiments of tumor cells and CAFs revealed that Prrx1-driven CAF-derived hepatocyte growth factor confers to a more invasive PDAC cell phenotype and resistant to therapy-induced apoptosis by inducing EMT in vitro. Importantly, in line with our in vitro and in vivo findings, compartment specific-gene expression analysis of human data revealed that pancreatic cancer patients with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC.
Conclusions: Here, we define that the Prrx1 transcription factor is critical for CAF plasticity, allowing a dynamic switch between different states. This work demonstrates that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
Citation Format: Karin Feldmann, Carlo Maurer, Katja Peschke, Steffen Teller, Kathleen Schuck, Katja Steiger, Thomas Engleitner, Rupert Öllinger, Aristeidis Papargyriou, Rim Sabrina Jahan Sarker, Wilko Weichert, Anil K. Rustgi, Roland M. Schmid, Roland Rad, Günter Schneider, Dieter Saur, Maximilian Reichert. Fibroblast plasticity driven by Prrx1 interferes the tumor cells - tumor microenvironment crosstalk towards a more aggressive pancreatic ductal adenocarcinoma abstract. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR003.
Abstract Cancer cells must develop strategies to adapt to the dynamically changing stresses caused by intrinsic or extrinsic processes, or therapeutic agents. Metabolic adaptability is crucial to ...mitigate such challenges. Considering metabolism as a central node of adaptability, it is focused on an energy sensor, the AMP‐activated protein kinase (AMPK). In a subtype of pancreatic ductal adenocarcinoma (PDAC) elevated AMPK expression and phosphorylation is identified. Using drug repurposing that combined screening experiments and chemoproteomic affinity profiling, it is identified and characterized PF‐3758309, initially developed as an inhibitor of PAK4, as an AMPK inhibitor. PF‐3758309 shows activity in pre‐clinical PDAC models, including primary patient‐derived organoids. Genetic loss‐of‐function experiments showed that AMPK limits the induction of ferroptosis, and consequently, PF‐3758309 treatment restores the sensitivity toward ferroptosis inducers. The work established a chemical scaffold for the development of specific AMPK‐targeting compounds and deciphered the framework for the development of AMPK inhibitor‐based combination therapies tailored for PDAC.
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